The present invention relates to devices and methods for cell culture, in particular of neuronal cells.
The brain is an extremely complex structure composed of several neuronal areas connected to one another. Experimental studies in vivo preserve this overall structure, but are not suitable for cell-scale study.
Cultures of disassociated cells make it possible to describe in much greater detail the system studied. For this reason, many laboratories perform neuronal cultures. Traditionally, these cultures of neurons are carried out in Petri dishes or culture wells. These cell cultures find applications as a reductionist model in the study of neurodegenerative diseases (Alzheimer's disease, Huntington's disease, Creutzfeldt-Jakob disease, etc.), but also in developmental biology for the understanding of molecular and cellular mechanisms of neuronal differentiation.
However, in these systems, the neuronal connections are made randomly and it is impossible to reconstitute therein an architecture similar to those that are found in vivo.
The network structure of the central nervous system (CNS) is completely absent, and does not make it possible to study how the various neuronal layers interact.
Another method consists in using slices of various parts of the brain, cultured ex vivo.
Even though the integrity of the neuronal layers is preserved by this method, the complexity of the tissues sampled quickly poses a problem. In order to understand more clearly the propagation of neuronal death and the mechanisms of development in the various layers of the brain, it is advisable to develop new experimental devices which make it possible to control the architecture of the networks of neurons cultured in vitro.
Microfluidics is a tool of choice for cell biology, and in particular for neurosciences.
In WO200434016, Jeon et al., inspired by the studies by Campenot [Campenot, R. B. Local control of neurite development by nerve growth factor, Proc. Natl. Acad. Sci. USA. 1977, 74(10), 4516-4519], propose a microfluidic circuit configuration which makes it possible to isolate the soma of neurons from their axon.
This configuration is suitable for the neurons of the central nervous system (CNS).
The “somatic” compartment is the channel into which the freshly dissected neurons are introduced.
The distal channel is that toward which the axons head in passing through the microchannels. The soma of the neurons cannot pass through the microchannels. This is because the microchannels are too thin to allow the soma to pass through.
This device is a first step toward the control of CNS neuronal cultures in vitro. The diffusion times in the microchannels are long, which makes it possible to treat the distal and somatic compartments separately. The diffusion of what is contained in one of the compartments toward the other is compensated for by imposing a pressure differential. For this, it is sufficient to place a larger volume of liquid in one of the reservoirs of one of the compartments, so as to impose a hydrostatic pressure differential between different compartments.
However, this device also has many limitations. Firstly, while it makes it possible to separate the cell compartments, it does not make it possible to induce any directed axonal connection between two neuron populations, since the axons can travel through the microchannels in both directions.
The publications WO 2006/037033 and U.S. Pat. No. 7,419,822, which are incorporated by way of reference, also relate to cell culture devices suitable for the culture of neurons.
The present invention aims, inter alia, to remedy these various limitations, and to thus allow studies, methods and screenings that are impossible with the current prior art devices.